Many techniques in Molecular Biology, Biochemistry and Chemistry rely upon the process of precipitation. There are two types of precipitation.
In the first type of precipitation, the components from a complex solution that are not of interest are selectively precipitated. The precipitate and supernatant are then separated (usually by centrifugation or filtration) and the supernatant is kept for further use.
In the second type of precipitation, the components of interest from a complex solution are selectively precipitated. The precipitate and supernatant are separated (again by centrifugation or filtration) and the precipitate is kept for further use. This precipitate may well be redissolved for further use.
Examples of precipitation that are of particular relevance to this invention will now be discussed.
a. Alcohol Precipitation of Nucleic Acid Molecules from Solution:
Alcohol precipitation of nucleic acid molecules from solution is a standard procedure for the concentration and/or purification of these species from complex solutions. Typical methods involve the addition of salt (e.g. 0.1 volumes of 2.5M sodium acetate (pH 5.2)) to a solution containing nucleic acids followed by addition of an alcohol (e.g. 2.5 volumes of ethanol). The nucleic acids then precipitate. The precipitated nucleic acid molecules aggregate (usually with the aid of reduced temperatures; e.g. 5 minutes on dry ice) and are recovered by centrifugation. After removal of the supernatant, the pelleted precipitate is normally redissolved in the required volume of an appropriate buffer. The nucleic acid may be DNA (partially or wholly single or double stranded), RNA (partially or wholly single or double stranded), mixtures of any of the above or a hybrid RNA/DNA species. The salt used may be sodium acetate, sodium chloride, potassium acetate, potassium chloride, ammonium acetate, ammonium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, guanidinium chloride or mixtures of the above. The alcohol used is normally ethanol or isopropanol.
b. Precipitation of Bacteriophage and Other Viruses from Solution:
Precipitation of bacteriophage and other viruses from solution by the addition of solutions containing high concentrations of highly hydratable polymers, such as polyethylene glycol (PEG), and salts, such as sodium chloride, is a standard procedure for the concentration and/or purification of these species from complex solutions. The bacteriophage or other viruses precipitated in this way may be used for nucleic acid extraction, protein extraction, infection of host cells, structural studies or immunological studies. A typical procedure involves the addition of 0.2 volumes of 20% (w/v) PEG in 2.5M sodium chloride to the complex solution known to contain the bacteriophage or other viruses. The bacteriophage or other viruses precipitate. The precipitated particles then aggregate (normally with the aid of incubation at reduced temperatures; e.g. 60 minutes at 4.degree. C.) and are recovered by centrifugation. After removal of the supernatant, the pellet (comprising precipitated particles of bacteriophage or other viruses) is normally redissolved in the required volume of an appropriate buffer. The bacteriophage may be filamentous (e.g. M13) or complex (e.g. lambda). They may infect bacteria, animal or plant cells and they may be DNA-containing or RNA-containing.
c. Removal of Bacterial DNA, Proteins and Membranes from Bacterial Lysates:
Another type of precipitation of interest to Molecular Biologists is used for the removal of bacterial DNA, proteins and membranes from bacterial lysates containing, in addition to the above, RNA and plasmid DNA and/or cosmid DNA and/or bacteriophage DNA. This forms the basis of the alkaline lysis procedure for preparations of low molecular weight DNA. In this procedure, the bacterial cells (e.g. E. coli) are lysed by treatment with sodium hydroxide (e.g. 200 mM) and the detergent sodium dodecyl sulphate (SDS) (e.g. 0.3-1.0% (w/v)). Addition of a mixture of either sodium or potassium acetate at low pH (e.g. 0.5 times the volume of lysis buffer of 3M sodium or potassium acetate adjusted to pH 4.8 with acetic acid) leads to the formation of a precipitate containing protein, membrane fragments and the entrapped bacterial DNA. The RNA and low molecular weight DNA species are not entrapped in this precipitate and can be recovered from the supernatant after centrifugation or filtration of the precipitate. The low molecular weight DNA species can be purified and/or concentrated, along with cellular RNA, by subsequent alcohol precipitation from this supernatant as described above. The DNA species extracted by this procedure may be plasmid, cosmid or bacteriophage-derived. The volume of cells lysed can be as little as a few microliters or as large as many liters of bacterial culture.